The capacity of a cell to alter its morphology and migrate is inherent to cancer cell metastasis. Although the precise biological mechanisms shaping cellular morphology during metastasis have not been elucidated, it is known that such changes involve cell-matrix interactions and cytoskeletal elements. The involvement of Na+ channels in shaping cellular morphology has been described for neurons (Mattei et al., Journal of Neuroscience Research, 55(6):666-73, 1999). As of yet, the intracellular mechanisms through which Na+ channel activity regulate cancer cellular morphology are unclear, although ion channels have been implicated in several types of cellular behavior that could be related to the different stages of metastasis. These include proliferation, migration, and adhesion/interaction with the cellular matrix.
Voltage-gated ion channels, classically associated with impulse conduction in excitable tissues, are also found in a variety of epithelial cell types where their function is not well known. Nine mammalian sodium channel genes have been identified and found to be expressed and functional. These genes are greater than 50% identical in amino acid sequence in the transmembrane and extracellular domains. Recently, several types of voltage-gated ion channels have been discovered in rat and human prostate cancer cells. Several independent studies have also linked a prostate voltage gated sodium (Na+) channel α-subunit with the invasiveness of human prostate cell lines including LNCaP and PC-3 (see Diss, et al., The Prostate, 48:165-178, 2001 and Smith et al., FEBS Letters, 423:19-24, 1998.). Further, electrophysiological studies using a whole-ell patch clamp indicated that the identified prostate cell sodium channel is sensitive to tetrodotoxin (TTX) at 600 nM, identifying the channel as voltage dependent TTX sensitive Na+ channel protein.
Comparisons between rodent and human prostate cancer cell lines led to the conclusion that the level of Na+ channel expression is associated positively with the invasiveness of prostate cancer cells in vitro. Encouragingly, both protein and functional studies strongly support sodium channel blockade as a viable mechanism for PCa cell inhibition. Recently, the effect of four anticonvulsants on the secretion of prostate-specific antigen (PSA) and interleukin-6 (IL-6) by human prostate cancer cell lines (LNCaP, DU-145 and PC-3) was measured using ELISA's specific for each protein. The results demonstrated that both phenytoin and carbamazepine, which inactivate voltage-gated sodium channels (NVSC), inhibit the secretion of PSA by LNCaP and IL-6, DU-145 and PC-3 cell lines (Abdul, M. and Hoosein, N., Anticancer Research, 21(3B):2045-8, 2001 May-June). Additionally, the authors demonstrate a reduced capacity to form colonies in Matrigel upon treatment with phenytoin. These data indicate further that sodium channel blockade is a strong candidate for effective treatment of PCa.
Experiments using tritiated batrachotoxin (BTX) have revealed an allosteric relationship between BTX and the phenytoin binding site in brain tissue. This relationship led applicant to investigate the neuronal hydantoin receptor in the brain for conformation and lipophilic properties. Since there was little structural data about the phenytoin-binding site on the NVSC, a defined series of compounds was designed, synthesized and evaluated to identify novel Na+ channel blocking agents. Such compounds have utility in treating diseases associated with hyper sodium channel activity, including treating epilepsy, pain, bipolar disease, depression Amytrophic lateral sclerosis (ALS) and neoplastic disease such as androgen-sensitive and androgen-independent prostate cancer.
Prostate neoplasia is the most common cause of cancer in men and the second leading cause of cancer death among men in the U.S. Approximately 189,000 men will be diagnosed with prostate cancer and approximately 30,000 will die from this disease in 2002. Human prostate cancer cells express a voltage gated sodium channel, a 260 Kd transmembrane protein that is similar to neuronal subtypes. Whole cell patch clamping experiments indicate that the prostate voltage sodium channel (PVSC) also functions similarly to neuronal subtypes. Significantly, Na+ channel expression in prostate cancer cells has been correlated positively to invasiveness in the highly metastatic cell line MAT-LyLu (rat). PVSC has been found to be sensitive to Tetrodotoxin (TTX) and it has been reported that TTX inhibits the invasiveness of PC-3 cells (human) by 31% (P=0.02) Laniado, et al. American Journal of Pathology, 150(4): 1213-21, 1997. Furthermore, TTX (6 mM) produces alterations in prostate cancer cell morphology, including a decrease in cell process length, field diameter; increases in cell body diameter and process thickness. S. P. Fraser, Y. Ding, A. Liu, C. S. Foster M. B. A. Djamgoz. Cell Tissue Research. 295: 505-512, 1999 and Grimes J A. Djamgoz M B. Journal of Cellular Physiology. 175(1):50-8, 1998. Therefore, PVSC serves as an effective target for potential prostate cancer therapeutics, thus presenting a need for new inhibitors of this sodium channel.